Selection of antigenic variants of the S glycoprotein of feline infectious peritonitis virus and analysis of antigenic sites involved in neutralization.
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The type II feline infectious peritonitis virus (FIPV) epitopes for neutralizing and enhancing antibodies are present on large spike glycoprotein (S) protein. In this study, we established monoclonal antibody-resistant mutant viruses resistant to three different monoclonal antibodies with neutralizing activity in Felis catus whole fetus cells and enhancing activity in feline macrophages, recognizing distinct epitopes on type II FIPV S protein. By comparing the nucleotide sequences of these mutant viruses with that of wild-type virus, we attempted to identify the neutralizing epitopes. The mutations were localized in the region of amino acid residues from 480 to 649 from the N terminal of the S protein. (+info)
Retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier.
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Coronaviruses generally have a narrow host range, infecting one or just a few species. Using targeted RNA recombination, we constructed a mutant of the coronavirus mouse hepatitis virus (MHV) in which the ectodomain of the spike glycoprotein (S) was replaced with the highly divergent ectodomain of the S protein of feline infectious peritonitis virus. The resulting chimeric virus, designated fMHV, acquired the ability to infect feline cells and simultaneously lost the ability to infect murine cells in tissue culture. This reciprocal switch of species specificity strongly supports the notion that coronavirus host cell range is determined primarily at the level of interactions between the S protein and the virus receptor. The isolation of fMHV allowed the localization of the region responsible for S protein incorporation into virions to the carboxy-terminal 64 of the 1,324 residues of this protein. This establishes a basis for further definition of elements involved in virion assembly. In addition, fMHV is potentially the ideal recipient virus for carrying out reverse genetics of MHV by targeted RNA recombination, since it presents the possibility of selecting recombinants, no matter how defective, that have regained the ability to replicate in murine cells. (+info)
Assembly of spikes into coronavirus particles is mediated by the carboxy-terminal domain of the spike protein.
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The type I glycoprotein S of coronavirus, trimers of which constitute the typical viral spikes, is assembled into virions through noncovalent interactions with the M protein. Here we demonstrate that incorporation is mediated by the short carboxy-terminal segment comprising the transmembrane and endodomain. To this aim, we used the virus-like particle (VLP) system that we developed earlier for the mouse hepatitis virus strain A59 (MHV-A59) and which we describe now also for the unrelated coronavirus feline infectious peritonitis virus (FIPV; strain 79-1146). Two chimeric MHV-FIPV S proteins were constructed, consisting of the ectodomain of the one virus and the transmembrane and endodomain of the other. These proteins were tested for their incorporation into VLPs of either species. They were found to assemble only into viral particles of the species from which their carboxy-terminal domain originated. Thus, the 64-terminal-residue sequence suffices to draw the 1308 (MHV)- or 1433 (FIPV)-amino-acid-long mature S protein into VLPs. Both chimeric S proteins appeared to cause cell fusion when expressed individually, suggesting that they were biologically fully active. This was indeed confirmed by incorporating one of the proteins into virions which thereby acquired a new host cell tropism, as will be reported elsewhere. (+info)
Molecular determinants of species specificity in the coronavirus receptor aminopeptidase N (CD13): influence of N-linked glycosylation.
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Aminopeptidase N (APN), a 150-kDa metalloprotease also called CD13, serves as a receptor for serologically related coronaviruses of humans (human coronavirus 229E [HCoV-229E]), pigs, and cats. These virus-receptor interactions can be highly species specific; for example, the human coronavirus can use human APN (hAPN) but not porcine APN (pAPN) as its cellular receptor, and porcine coronaviruses can use pAPN but not hAPN. Substitution of pAPN amino acids 283 to 290 into hAPN for the corresponding amino acids 288 to 295 introduced an N-glycosylation sequon at amino acids 291 to 293 that blocked HCoV-229E receptor activity of hAPN. Substitution of two amino acids that inserted an N-glycosylation site at amino acid 291 also resulted in a mutant hAPN that lacked receptor activity because it failed to bind HCoV-229E. Single amino acid revertants that removed this sequon at amino acids 291 to 293 but had one or five pAPN amino acid substitution(s) in this region all regained HCoV-229E binding and receptor activities. To determine if other N-linked glycosylation differences between hAPN, feline APN (fAPN), and pAPN account for receptor specificity of pig and cat coronaviruses, a mutant hAPN protein that, like fAPN and pAPN, lacked a glycosylation sequon at 818 to 820 was studied. This sequon is within the region that determines receptor activity for porcine and feline coronaviruses. Mutant hAPN lacking the sequon at amino acids 818 to 820 maintained HCoV-229E receptor activity but did not gain receptor activity for porcine or feline coronaviruses. Thus, certain differences in glycosylation between coronavirus receptors from different species are critical determinants in the species specificity of infection. (+info)
Adverse effects of feline IL-12 during DNA vaccination against feline infectious peritonitis virus.
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Cell-mediated immunity is thought to play a decisive role in protecting cats against feline infectious peritonitis (FIP), a progressive and lethal coronavirus disease. In view of the potential of DNA vaccines to induce cell-mediated responses, their efficacy to induce protective immunity in cats was evaluated. The membrane (M) and nucleocapsid (N) proteins were chosen as antigens, because antibodies to the spike (S) protein of FIP virus (FIPV) are known to precipitate pathogenesis. However, vaccination by repeated injections of plasmids encoding these proteins did not protect kittens against challenge infection with FIPV. Also, a prime-boost protocol failed to afford protection, with priming using plasmid DNA and boosting using recombinant vaccinia viruses expressing the same coronavirus proteins. Because of the role of IL-12 in initiating cell-mediated immunity, the effects of co-delivery of plasmids encoding the feline cytokine were studied. Again, IL-12 did not meet expectations - on the contrary, it enhanced susceptibility to FIPV challenge. This study shows that DNA vaccination failed to protect cats against FIP and that IL-12 may yield adverse effects when used as a cytokine adjuvant. (+info)
Mutational analysis of the active centre of coronavirus 3C-like proteases.
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Formation of the coronavirus replication-transcription complex involves the synthesis of large polyprotein precursors that are extensively processed by virus-encoded cysteine proteases. In this study, the coding sequence of the feline infectious peritonitis virus (FIPV) main protease, 3CL(pro), was determined. Comparative sequence analyses revealed that FIPV 3CL(pro) and other coronavirus main proteases are related most closely to the 3C-like proteases of potyviruses. The predicted active centre of the coronavirus enzymes has accepted unique replacements that were probed by extensive mutational analysis. The wild-type FIPV 3CL(pro) domain and 25 mutants were expressed in Escherichia coli and tested for proteolytic activity in a peptide-based assay. The data strongly suggest that, first, the FIPV 3CL(pro) catalytic system employs His(41) and Cys(144) as the principal catalytic residues. Second, the amino acids Tyr(160) and His(162), which are part of the conserved sequence signature Tyr(160)-Met(161)-His(162) and are believed to be involved in substrate recognition, were found to be indispensable for proteolytic activity. Third, replacements of Gly(83) and Asn(64), which were candidates to occupy the position spatially equivalent to that of the catalytic Asp residue of chymotrypsin-like proteases, resulted in proteolytically active proteins. Surprisingly, some of the Asn(64) mutants even exhibited strongly increased activities. Similar results were obtained for human coronavirus (HCoV) 3CL(pro) mutants in which the equivalent Asn residue (HCoV 3CL(pro) Asn(64)) was substituted. These data lead us to conclude that both the catalytic systems and substrate-binding pockets of coronavirus main proteases differ from those of other RNA virus 3C and 3C-like proteases. (+info)
Detection of feline coronavirus in captive Felidae in the USA.
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Feline coronavirus (FCoV) is an important pathogen of domestic and nondomestic Felidae. Investigation into the prevalence of FCoV in exotic Felidae has relied primarily on serology. The usefulness of genetic detection of FCoV using reverse transcription and nested polymerase chain reaction (RT/nPCR) for viral screening was investigated. Seventy-five biologic samples, primarily feces, from captive felids from 11 institutions were tested using PCR. Serum samples collected from all but 12 of these animals were tested for antibodies to type I and type II FCoV by indirect immunofluorescence. Twenty-four animals were positive using RT/nPCR for virus. Twenty-nine animals were seropositive to type I and/or type II FCoV. From serologic data, infection with a virus antigenically related to FCoV type I occurred most commonly. Serology did not correlate with virus shedding because 13 animals were seronegative to FCoV type I and II but positive using RT/nPCR for virus. Conversely, 20 animals were seropositive but negative using RT/nPCR for FCoV. Some of the populations in which virus was detected had experienced health problems, including feline infectious peritonitis (FIP), necrotizing colitis, and mild enteritis. In addition to its role in FIP, this virus may play a role in gastrointestinal diseases of infected animals. This study demonstrates that FCoV is a significant infectious agent of captive felids because over half of the animals tested were positive by viral genetic detection, serology, or both. Dependence upon one method for detection of infection is unreliable. (+info)
Switching species tropism: an effective way to manipulate the feline coronavirus genome.
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Feline infectious peritonitis virus (FIPV), a coronavirus, is the causative agent of an invariably lethal infection in cats. Like other coronaviruses, FIPV contains an extremely large positive-strand RNA genome of ca. 30 kb. We describe here the development and use of a reverse genetics strategy for FIPV based on targeted RNA recombination that is analogous to what has been described for the mouse hepatitis virus (MHV) (L. Kuo et al., J. Virol. 74:1393-1406, 2000). In this two-step process, we first constructed by targeted recombination a mutant of FIPV, designated mFIPV, in which the ectodomain of the spike glycoprotein was replaced by that of MHV. This switch allowed for the selection of the recombinant virus in murine cells: mFIPV grows to high titers in these cells but has lost the ability to grow in feline cells. In a second, reverse process, mFIPV was used as the recipient, and the reintroduction of the FIPV spike now allowed for selection of candidate recombinants by their regained ability to grow in feline cells. In this fashion, we reconstructed a wild-type recombinant virus (r-wtFIPV) and generated a directed mutant FIPV in which the initiation codon of the nonstructural gene 7b had been disrupted (FIPV Delta 7b). The r-wtFIPV was indistinguishable from its parental virus FIPV 79-1146 not only for its growth characteristics in tissue culture but also in cats, exhibiting a highly lethal phenotype. FIPV Delta 7b had lost the expression of its 7b gene but grew unimpaired in cell culture, confirming that the 7b glycoprotein is not required in vitro. We establish the second targeted RNA recombination system for coronaviruses and provide a powerful tool for the genetic engineering of the FIPV genome. (+info)